Dock-and-Lock (DNL) Complexes for Delivery of Interference RNA

ABSTRACT

Described herein are compositions and methods of use of targeted delivery complexes for delivery of siRNA to a disease-associated cell, tissue or pathogen. The targeted delivery complex comprises a targeting molecule, such as an antibody or fragment thereof, conjugated to one or more siRNA carriers. In preferred embodiments the siRNA carrier is a dendrimer or protamine and the targeting molecule is an anti-cancer antibody, such as hRS7. More preferably, the antibody or fragment is rapidly internalized into the target cell to facilitate uptake of the siRNA. Most preferably, the targeted delivery complex is made by the DNL technique. The compositions and methods are of use to treat a variety of disease states, such as cancer, autoimmune disease, immune dysfunction, cardiac disease, neurologic disease, inflammatory disease or infectious disease.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. Nos. 12/949,536, filed Nov. 18, 2010, (which was a divisional ofSer. No. 12/396,605, filed Mar. 3, 2009, which was a divisional of U.S.Pat. No. 7,527,787); Ser. No. 12/915,515, filed Oct. 29, 2010; Ser. No.12/871,345, filed Aug. 30, 2010; Ser. No. 12/869,823, filed Aug. 27,2010; Ser. No. 12/754,740, filed Apr. 6, 2010; Ser. No. 12/752,649,filed Apr. 1, 2010; Ser. No. 12/731,781, filed Mar. 25, 2010; Ser. No.12/644,146 (which was a divisional of U.S. Pat. No. 7,666,400), filedDec. 22, 2009; Ser. No. 12/544,476, filed Aug. 20, 2009; Ser. No.12/468,589 (which was a divisional of U.S. Pat. No. 7,550,143), filedMay 19, 2009; Ser. No. 12/418,877, filed Apr. 6, 2009; Ser. No.12/417,917 (which was a divisional of U.S. Pat. No. 7,534,866), filedApr. 3, 2009; and Ser. No. 12/396,965 (which was a divisional of U.S.Pat. No. 7,521,056), filed Mar. 3, 2009. Those applications claimed thebenefit under 35 U.S.C. 119(e) of provisional U.S. Patent Applications61/378,059, filed Aug. 30, 2010; 61/323,960, filed Apr. 14, 2010;61/316,996, filed Mar. 24, 2010; 61/266,305, filed Dec. 3, 2009;61/258,729, filed Nov. 6, 2009; 61/258,369, filed Nov. 5, 2009;61/238,424, filed Aug. 31, 2009; 61/238,473, filed Aug. 31, 2009;61/168,715, filed Apr. 13, 2009; 61/168,668, filed Apr. 13, 2009;61/168,657, filed Apr. 13, 2009; 61/168,290, filed Apr. 10, 2009;61/166,809, filed Apr. 6, 2009; 61/163,666, filed Mar. 26, 2009;61/119,542, filed Dec. 3, 2008; 61/104,916, filed Oct. 13, 2008;61/090,487, filed Aug. 20, 2008; 61/043,932, filed Apr. 6, 2008;60/864,530, filed Nov. 6, 2006; 60/782,332, filed Mar. 14, 2006;60/751,196, filed Dec. 16, 2005; 60/728,292, filed Oct. 19, 2005;60/668,603, filed Apr. 6, 2005; and 60/544,227, filed Feb. 13, 2004.

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application Ser. Nos. 61/267,877, filed Dec. 9, 2009;61/302,682, filed Feb. 8, 2010 and 61/414,592, filed Nov. 17, 2010. Thetext of each priority application is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention concerns compositions and methods for targeted deliveryof RNAi species, such as siRNA. Preferably, the targeted deliverycomplexes comprise an antibody, antibody fragment or other targetingmolecule conjugated to a carrier molecule for siRNA, such as a protamineor a dendrimer. More preferably, the targeted delivery complexes aremade by the dock-and-lock (DNL) technique. The targeted delivery complexmay comprise one or more siRNA species for delivery to a targeted cellor tissue. Most preferably, delivery of siRNA is effective to treat adisease, syndrome or disorder, such as cancer, autoimmune disease,immune dysfunction (e.g., graft-versus-host disease or organ transplantrejection), inflammation, infectious disease, cardiovascular disease,endocrine or metabolic disease or neurodegenerative disease. For diseasetherapy, the antibody or other targeting molecule binds to a targetantigen produced by a diseased cell or tissue or otherwise associatedwith the disease state. The DNL complex may comprise multiple copies ofa carrier molecule to efficiently deliver therapeutic siRNA to a targetcell. The antibody or other targeting molecule may be a rapidlyinternalizing species to facilitate intracellular uptake of siRNA.

2. Related Art

RNA interference (RNAi) is a naturally occurring regulatory mechanismfor control of gene expression in cells (Fire et al., 1998, Nature391:806-11). RNAi is mediated by the RNA-induced silencing complex(RISC) and is initiated by short double-stranded RNA molecules thatinteract with the catalytic RISC component argonaute (Rand et al., 2005,Cell 123:621-29). Types of RNAi molecules include microRNA (miRNA) andsmall interfering RNA (siRNA). RNAi species can bind with messenger RNA(mRNA) through complementary base-pairing and inhibits gene expressionby post-transcriptional gene silencing. Upon binding to a complementarymRNA species, RNAi induces cleavage of the mRNA molecule by theargonaute component of RISC. Among other characteristics, miRNA andsiRNA differ in the degree of specificity for particular gene targets,with siRNA being relatively specific for a particular target gene andmiRNA inhibiting translation of multiple mRNA species.

Therapeutic use of RNAi by inhibition of selected gene expression hasbeen attempted for a variety of disease states, such as maculardegeneration and respiratory syncytial virus infection (Sah, 2006, LifeSci 79:1773-80). It has been suggested that siRNA functions in host celldefenses against viral infection and siRNA has been widely examined asan approach to anti-viral therapy (see, e.g., Zhang et al., 2004, NatureMed 11:56-62; Novina et al., 2002, Nature Med 8:681-86; Palliser et al.,2006, Nature 439:89-94). The use of siRNA for cancer therapy has alsobeen attempted. Fujii et al. (2006, Int J Oncol 29:541-48) transfectedHPV positive cervical cancer cells with siRNA against HPV E6 and E7 andsuppressed tumor growth. siRNA-mediated knockdown of metadherinexpression in breast cancer cells was reported to inhibit experimentallung metastasis (Brown and Ruoslahti, 2004, Cancer Cell 5:365-74).

Difficulties with siRNA-based therapies have included poor cellularuptake of exogenous siRNA and potential off-target effects on othergenes (see, e.g., Kim et al., 2009, Trends Molec Med 15:491-500).Jackson et al. (2003, Nat Biotechnol 21:635-37) designed differentsiRNAs targeting IGF-1R and MAPK14 and showed silencing of nontargetedgenes containing as few as eleven contiguous identical nucleotides tothe siRNA species.

Attempts have been made to provide targeted delivery of siRNA to reducethe potential for off-target toxicity. Song et al. (2005, Nat Biotechnol23:709-17) used protamine-conjugated Fab fragments against HIV envelopeprotein to deliver siRNA to circulating cells. Schiffelers et al. (2004,Nucl Acids Res 32:e149) conjugated RGD peptides to nanoparticles todeliver anti-VEGF R2 siRNA to tumors and inhibited tumor angiogenesisand growth rate in nude mice. Dickerson et al. used nanogelsfunctionalized with anti-EphA2 receptor peptides to chemosensitizeovarian cancer cells with siRNA against EGFR. Dendrimer-conjugatedmagnetic nanoparticles have been applied to the targeted delivery ofantisense survivin oligodeoxynucleotides (Pan et al., 2007, Cancer Res67:8156-63).

Despite recent progress, a need exists in the field for more effectivemeans of targeted delivery of therapeutic siRNA, to increase cellularuptake and minimize siRNA-related toxic side effects (Kim et al., 2009).

SUMMARY

The present invention concerns methods and compositions for efficientdelivery of siRNA species to disease-associated target cells, tissues orpathogens. Preferably, delivery is facilitated by use of targeteddelivery complexes, which comprise an antibody, antibody fragment orother targeting molecule conjugated to a carrier molecule for siRNA,such as a protamine, dendrimer or nanoparticle. More preferably, thetargeting molecule provides for highly selective or specific delivery oftherapeutic siRNA to diseased cells or tissues. Most preferably, thetargeted delivery complex facilitates intracellular uptake of siRNA intotarget cells.

The siRNA species to be delivered and the corresponding specificity ofthe targeting molecule will be determined by the disease state to betreated. As discussed in more detail below, tens of thousands of siRNAsequences directed to mRNAs associated with a broad variety of diseasestates are known in the art and may be obtained from public databases,such as siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. Many siRNA species may alsobe purchased from commercial vendors like Sigma-Aldrich (St Louis, Mo.),Invitrogen (Carlsbad, Calif.), Santa Cruz Biotechnology (Santa Cruz,Calif.), Ambion (Austin, Tex.), Dharmacon (Thermo Scientific, Lafayette,Colo.), Promega (Madison, Wis.), Minis Bio (Madison, Wis.) and Qiagen(Valencia, Calif.).

A variety of carrier moieties for siRNA have been reported and any suchknown carrier may be incorporated into a targeted delivery complex foruse. Non-limiting examples of carriers include protamine (Rossi, 2005,Nat Biotech 23:682-84; Song et al., 2005, Nat Biotech 23:709-17);dendrimers such as PAMAM dendrimers (Pan et al., 2007, Cancer Res.67:8156-8163); polyethylenimine (Schiffelers et al., 2004, Nucl AcidsRes 32:e149); polypropyleneimine (Taratula et al., 2009, J ControlRelease 140:284-93); polylysine (Inoue et al., 2008, J Control Release126:59-66); histidine-containing reducible polycations (Stevenson etal., 2008, J Control Release 130:46-56); histone H1 protein (Haberlandet al., 2009, Mol Biol Rep 26:1083-93); cationic comb-type copolymers(Sato et al., 2007, J Control Release 122:209-16); polymeric micelles(U.S. Patent Application Publ. No. 20100121043); and chitosan-thiaminepyrophosphate (Rojanarata et al., 2008, Pharm Res 25:2807-14). Theskilled artisan will realize that in general, polycationic proteins orpolymers are of use as siRNA carriers. The skilled artisan will furtherrealize that siRNA carriers can also be used to carry otheroligonucleotide or nucleic acid species, such as anti-senseoligonucleotides or short DNA genes.

Various embodiments may concern use of the targeted delivery complexesto treat a disease, including but not limited to non-Hodgkin'slymphomas, B-cell acute and chronic lymphoid leukemias, Burkittlymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronicmyeloid leukemias, T-cell lymphomas and leukemias, multiple myeloma,glioma, Waldenstrom's macroglobulinemia, carcinomas, melanomas,sarcomas, gliomas, and skin cancers. The carcinomas may be selected fromthe group consisting of carcinomas of the oral cavity, gastrointestinaltract, colon, stomach, pulmonary tract, lung, breast, ovary, prostate,uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver,gall bladder, kidney, skin, and testes.

In addition, the targeted delivery complexes may be used to treat anautoimmune disease, for example acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, or fibrosing alveolitis.

The compositions of the present invention also are useful for thetreatment of infections, where the antibody component of the targeteddelivery complex specifically binds to a disease-causing microorganism.In the context of the present invention a disease-causing microorganismincludes pathogenic bacteria, viruses, fungi and diverse parasites, andthe antibody can target these microorganisms, their products or antigensassociated with their lesions. Examples of microorganisms include, butare not limited to: Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Haemophilus influenzae B,Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis,Tetanus toxin, HIV-1, -2, -3, Hepatitis A, B, C, D, Rabies virus,Influenza virus, Cytomegalovirus, Herpes simplex I and II, Human serumparvo-like virus, Papilloma viruses, Polyoma virus, Respiratorysyncytial virus, Varicella-Zoster virus, Hepatitis B virus, Papillomavirus, Measles virus, Adenovirus, Human T-cell leukemia viruses,Epstein-Barr virus, Murine leukemia virus, Mumps virus, Vesicularstomatitis virus, Sindbis virus, Lymphocytic choriomeningitis virus,Wart virus, Blue tongue virus, Sendai virus, Feline leukemia virus,Reovirus, Polio virus, Simian virus 40, Mouse mammary tumor virus,Dengue virus, Rubella virus, protozoans, Plasmodium falciparum,Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosomacruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M.pneumoniae. Monoclonal antibodies that bind to these pathogenicmicroorganisms are well known in the art.

In one embodiment, a pharmaceutical composition of the present inventionmay be used to treat a subject having a metabolic disease, suchamyloidosis, or a neurodegenerative disease, such as Alzheimer'sdisease. Bapineuzumab is in clinical trials for Alzheimer's diseasetherapy. Other antibodies proposed for therapy of Alzheimer's diseaseinclude Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47),gantenerumab, and solanezumab. Infliximab, an anti-TNF-α antibody, hasbeen reported to reduce amyloid plaques and improve cognition. Anti-CD3antibodies have been proposed for therapy of type 1 diabetes (Cernea etal., 2010, Diabetes Metab Rev 26:602-05). In addition, a pharmaceuticalcomposition of the present invention may be used to treat a subjecthaving an immune-dysregulatory disorder such as graft-versus-hostdisease or organ transplant rejection.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD 15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL) and anti-CD74 (e.g., hLL1) antibodies can be used totarget atherosclerotic plaques. Abciximab (anti-glycoprotein IIb/IIIa)has been approved for adjuvant use for prevention of restenosis inpercutaneous coronary interventions and the treatment of unstable angina(Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3 antibodies havebeen reported to reduce development and progression of atherosclerosis(Steffens et al., 2006, Circulation 114:1977-84). Antibodies againstoxidized LDL induced a regression of established atherosclerosis in amouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1antibody was shown to reduce ischemic cell damage after cerebral arteryocclusion in rats (Zhang et al., 1994, Neurology 44:1747-51).Commercially available monoclonal antibodies to leukocyte antigens arerepresented by: OKT anti-T-cell monoclonal antibodies (available fromOrtho Pharmaceutical Company) which bind to normal T-lymphocytes; themonoclonal antibodies produced by the hybridomas having the ATCCaccession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 andG022 (Becton Dickinson); NEN9.4 (New England Nuclear); and FMC11 (SeraLabs). A description of antibodies against fibrin and platelet antigensis contained in Knight, Semin. Nucl. Med., 20:52-67 (1990).

Antibodies or other targeting molecules of use may bind to anydisease-associated antigen known in the art. Where the disease state iscancer, for example, many antigens expressed by or otherwise associatedwith tumor cells are known in the art, including but not limited to,carbonic anhydrase IX, alpha-fetoprotein, α-actinin-4, A3, antigenspecific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125,CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5,CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25,CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52,CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM,EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIT, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2,MUM-3, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody,placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA,PRAME, PSMA, P1GF, IGF, IGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE,5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors,TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complementfactors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6,Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensiet al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,54:187-207).

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785) and hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440) the Examples section of each cited patent or applicationincorporated herein by reference.

An antibody or antigen-binding fragment of use may be chimeric,humanized or human. The use of chimeric antibodies is preferred to theparent murine antibodies because they possess human antibody constantregion sequences and therefore do not elicit as strong a humananti-mouse antibody (HAMA) response as murine antibodies. The use ofhumanized antibodies is even more preferred, in order to further reducethe possibility of inducing a HAMA reaction. As discussed below,techniques for humanization of murine antibodies by replacing murineframework and constant region sequences with corresponding humanantibody framework and constant region sequences are well known in theart and have been applied to numerous murine anti-cancer antibodies.Antibody humanization may also involve the substitution of one or morehuman framework amino acid residues with the corresponding residues fromthe parent murine framework region sequences. As also discussed below,techniques for production of human antibodies are also well known.

In certain preferred embodiments the targeted delivery complex may beproduced using the dock-and-lock (DNL) technique (see, e.g., U.S. Pat.Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, theExamples section of each incorporated herein by reference). The DNLtechnique utilizes the specific binding interactions occurring between adimerization and docking domain (DDD moiety) from protein kinase A, andan anchoring domain (AD moiety) from any of a number of known A-kinaseanchoring proteins (AKAPs). The DDD moieties spontaneously form dimerswhich then bind to an AD moiety. By attaching appropriate effectormoieties, such as antibodies or fragments thereof and siRNA carriers, toAD and DDD moieties, the DNL technique allows the specific covalentformation of any desired targeted delivery complex. Where the effectormoiety is a protein or peptide, such as an antibody fragment, protamineor polylysine, the AD and DDD moieties may be incorporated into fusionproteins conjugated to the effector moieties. Where non-protein effectormoieties are utilized, chemical conjugation may be utilized to attachthe AD or DDD moiety.

In certain embodiments, disease therapy by targeted delivery of siRNAmay be enhanced by combination therapy with one or more othertherapeutic agents. Known therapeutic agents of use include toxins,immunomodulators (such as cytokines, lymphokines, chemokines, growthfactors and tumor necrosis factors), hormones, hormone antagonists,enzymes, oligonucleotides (such as siRNA or RNAi), photoactivetherapeutic agents, anti-angiogenic agents and pro-apoptotic agents. Thetherapeutic agents may be delivered by conjugation to the same ordifferent antibodies or other targeting molecules or may be administeredin unconjugated form. Other therapeutic agents may be administeredbefore, concurrently with or after the siRNA targeted delivery complex.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicins and theiranalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate, CPT-11, calicheamicinand a combination thereof.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof. Or animmunomodulator selected from the group consisting of a cytokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combinations thereof.

In other preferred embodiments, the therapeutic agent is a radionuclideselected from the group consisting of ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹¹At, ⁶⁷Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. Also preferred are radionuclides that substantiallydecay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m andIr-192. Decay energies of useful beta-particle-emitting nuclides arepreferably <1,000 keV, more preferably <100 keV, and most preferably <70keV. Also preferred are radionuclides that substantially decay withgeneration of alpha-particles. Such radionuclides include, but are notlimited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211,Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(133m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. In otherembodiments the therapeutic agent is a photoactive therapeutic agentselected from the group consisting of chromogens and dyes.

Alternatively, the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Such enzymes may be used, for example, incombination with prodrugs that are administered in relatively non-toxicform and converted at the target site by the enzyme into a cytotoxicagent. In other alternatives, a drug may be converted into less toxicform by endogenous enzymes in the subject but may be reconverted into acytotoxic form by the therapeutic enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Synthesis of E1-L-thP1 DNL complex for siRNA delivery. (A) Ananti-Trop-2 hRS7 antibody was produced with an AD2 moiety attached tothe C-terminal end of each antibody heavy chain. (B) A DDD2-L-thP1construct was synthesized with a truncated segment of human protamine 1and a DDD2 moiety attached respectively to the C- and N-terminal ends ofa humanized antibody light chain. (C) Incubation of the hRS7-IgG-AD2 andDDD2-L-thP1 under mild reducing conditions resulted in the formation ofthe protamine-conjugated hRS7 DNL complex, designated E1-L-thP1.

FIG. 2. Gel shift assay for DNA binding by E1-L-thP1.

FIG. 3. Internalization of siRNA mediated by E1-L-thP1 binding.

FIG. 4. Apoptosis induced by E1-L-thP1 internalization of siRNA.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the full-lengthantibody. The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions of antibodies, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”).

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB-cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which is incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent.

Interference RNA

The skilled artisan will realize that any siRNA or interference RNAspecies may be attached to the targeted delivery complexes for deliveryto a targeted tissue. Many siRNA species against a wide variety oftargets are known in the art, and any such known siRNA may be utilizedin the claimed methods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Mints Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Exemplary siRNA species known in the art are listed in Table 1. AlthoughsiRNA is delivered as a double-stranded molecule, for simplicity onlythe sense strand sequences are shown in Table 1.

TABLE 1 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 1 VEGF R2 AAGCTCAGCACACAGAAAGACSEQ ID NO: 2 CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO: 3 CXCR4GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 4 PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 5 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 6 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 7 E1A binding protein UGACACAGGCAGGCUUGACUUSEQ ID NO: 8 Plasminogen GGTGAAGAAGGGCGTCCAA SEQ ID NO: 9 activatorK-ras GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO: 10CAAGAGACTCGCCAACAGCTCCAACT TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAGSEQ ID NO: 11 Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 12Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 13 Bcl-XUAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 14 Raf-1 TTTGAATATCTGTGCTGAGAACACASEQ ID NO: 15 GTTCTCAGCACAGATATTCTTTTT Heat shockAATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID NO: 16 transcription factor 2IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 17 ThioredoxinAUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 18 CD44 GAACGAAUCCUGAAGACAUCUSEQ ID NO: 19 MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 20 MAPKAPK2UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 21 FGFR1 AAGTCGGACGCAACAGAGAAASEQ ID NO: 22 ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO: 23 BCL2L1CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 24 ABL1 TTAUUCCUUCUUCGGGAAGUCSEQ ID NO: 25 CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO: 26 CD9GAGCATCTTCGAGCAAGAA SEQ ID NO: 27 CD151 CATGTGGCACCGTTTGCCTSEQ ID NO: 28 Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID NO: 29 BRCA1UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 30 p53 GCAUGAACCGGAGGCCCAUTTSEQ ID NO: 31 CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO: 32

The skilled artisan will realize that Table 1 represents a very smallsampling of the total number of siRNA species known in the art, and thatany such known siRNA may be utilized in the claimed methods andcompositions.

Antibody Preparation

In certain embodiments, the targeted delivery complex may comprise amonoclonal antibody (MAb) or antigen-binding antibody fragment. MAbs canbe isolated and purified from hybridoma cultures by a variety ofwell-established techniques. Such isolation techniques include affinitychromatography with Protein-A or Protein-G Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992). After theinitial raising of antibodies to the immunogen, the antibodies can besequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies and Target Antigens

As discussed above, in preferred embodiments the targeted deliverycomplexes are of use for treatment of malignant disease, cardiovasculardisease, infectious disease, inflammatory disease, autoimmune disease,metabolic disease, immune dysfunction disease (e.g., graft versus hostdisease or organ transplant rejection) or neurological (e.g.,neurodegenerative) disease. Exemplary target antigens of use fortreating such diseases may include carbonic anhydrase IX, CCCL19,CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1α, AFP, PSMA,CEACAM5, CEACAM6, c-met, B7, ED-B of fibronectin, Factor H, FHL-1,Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1 (IGF-1), IFN-γ, IFN-α, IFN-β, IL-2,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15,IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF,MUC1, MUC2, MUC3, MUC4, MUC5, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2,HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAILreceptor (R1 and R2), VEGFR, EGFR, P1GF, complement factors C3, C3a,C3b, C5a, C5, PLAGL2, and an oncogene product.

In certain embodiments, such as treating tumors, antibodies of use maytarget tumor-associated antigens. These antigenic markers may besubstances produced by a tumor or may be substances which accumulate ata tumor site, on tumor cell surfaces or within tumor cells. Among suchtumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744, the Examplessection of each of which is incorporated herein by reference. Reports ontumor associated antigens (TAAs) include Mizukami et al., (2005, NatureMed. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Renet al. (2005, Ann. Surg. 242:55-63), each incorporated herein byreference with respect to the TAAs identified.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256 (2000).Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma). TACIand B-cell maturation antigen (BCMA) are bound by the tumor necrosisfactor homolog—a proliferation-inducing ligand (APRIL). APRIL stimulatesin vitro proliferation of primary B and T-cells and increases spleenweight due to accumulation of B-cells in vivo. APRIL also competes withTALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA andTACI specifically prevent binding of APRIL and block APRIL-stimulatedproliferation of primary B-cells. BCMA-Fc also inhibits production ofantibodies against keyhole limpet hemocyanin and Pneumovax in mice,indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI arerequired for generation of humoral immunity. Thus, APRIL-TALL-I andBCMA-TACI form a two ligand-two receptor pathway involved in stimulationof B and T-cell function.

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138,CD154, CXCR4, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, P1GF,ED-B fibronectin, an oncogene (e.g., c-met or PLAGL2), an oncogeneproduct, CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF,TRAIL-R1 (DR4) and TRAIL-R2 (DR5).

The skilled artisan will realize that any antibody or fragment known inthe art that has binding specificity for a target antigen associatedwith a disease state or condition may be utilized. Such known antibodiesinclude, but are not limited to, hR1 (anti-IGF-1R, U.S. patentapplication Ser. No. 12/772,645, filed Mar. 12, 2010) hPAM4(anti-pancreatic cancer mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,662,378, U.S.patent application Ser. No. 12/846,062, filed Jul. 29, 2010), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496)the Examples section of each cited patent or application incorporatedherein by reference.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.)

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,15; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 Kdfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulthydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The V_(κ) sequence for the MAb may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the V_(κ) andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the V_(κ) andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Dock-and-Lock (DNL)

In certain embodiments, targeted delivery complexes may be producedusing the dock-and-lock technology (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examples section ofeach incorporated herein by reference). The DNL method exploits specificprotein/protein interactions that occur between the regulatory (R)subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain(AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₁b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNLconstructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site—specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Immunoconjugates

In preferred embodiments, a moiety such as a siRNA carrier moiety may becovalently attached to an antibody or antibody fragment to form animmunoconjugate. Carrier moieties may be attached, for example toreduced SH groups and/or to carbohydrate side chains. A carrier moietycan be attached at the hinge region of a reduced antibody component viadisulfide bond formation. Alternatively, such agents can be attachedusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted akyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,comprising administering a therapeutically effective amount of atargeted delivery complex, such as an antibody conjugated to a siRNAcarrier and loaded with siRNA moieties.

The targeted delivery complexes can be supplemented with theadministration, either concurrently or sequentially, of at least oneother therapeutic agent. Multimodal therapies may include therapy withother antibodies, such as anti-CD22, anti-CD19, anti-CD20, anti-CD21,anti-CD74, anti-CD80, anti-CD23, anti-CD45, anti-CD46, anti-MIF,anti-EGP-1, anti-CEACAM5, anti-CEACAM6, anti-pancreatic cancer mucin,anti-IGF-1R or anti-HLA-DR (including the invariant chain) antibodies inthe form of naked antibodies, fusion proteins, or as immunoconjugates.Various antibodies of use, such as anti-CD19, anti-CD20, and anti-CD22antibodies, are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353(1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393; 6,676,924;7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786;7,282,567; 7,300,655; 7,312,318; 7,612,180; 7,501,498; the Examplessection of each of which is incorporated herein by reference.

In another form of multimodal therapy, subjects receive targeteddelivery complexes in conjunction with standard cancer chemotherapy. Forexample, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and150-200 mg/m² carmustine) is a regimen used to treat non-Hodgkin'slymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well-known to those of skillin the art. See, for example, Freedman et al., “Non-Hodgkin'sLymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.(eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, firstgeneration chemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

In a preferred multimodal therapy, both chemotherapeutic drugs andcytokines are co-administered with a targeted delivery complex. Thecytokines, chemotherapeutic drugs and targeted delivery complex can beadministered in any order, or together.

Targeted delivery complexes can be formulated according to known methodsto prepare pharmaceutically useful compositions, whereby the targeteddelivery complex is combined in a mixture with a pharmaceuticallysuitable excipient. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The targeted delivery complex of the present invention can be formulatedfor intravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the targeted delivery complex isinfused over a period of less than about 4 hours, and more preferably,over a period of less than about 3 hours. For example, the first 25-50mg could be infused within 30 minutes, preferably even 15 min, and theremainder infused over the next 2-3 hrs. Formulations for injection canbe presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The targeted delivery complex may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the targeted delivery complex is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours.

More generally, the dosage of an administered targeted delivery complexfor humans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage oftargeted delivery complex that is in the range of from about 1 mg/kg to25 mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. A dosage of1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or 41-824mg/m² for a 1.7-m patient. The dosage may be repeated as needed, forexample, once per week for 4-10 weeks, once per week for 8 weeks, oronce per week for 4 weeks. It may also be given less frequently, such asevery other week for several months, or monthly or quarterly for manymonths, as needed in a maintenance therapy.

Alternatively, a targeted delivery complex may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, the targeted delivery complex may be administered twice per week for4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m² (340mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), itmay be administered once or even twice weekly for 4 to 10 weeks.Alternatively, the dosage schedule may be decreased, namely every 2 or 3weeks for 2-3 months. It has been determined, however, that even higherdoses, such as 20 mg/kg once weekly or once every 2-3 weeks can beadministered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the subject targeted delivery complexes are ofuse for therapy of cancer. Examples of cancers include, but are notlimited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, andleukemia, myeloma, or lymphoid malignancies. More particular examples ofsuch cancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Other Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially with the targeted delivery complex. For example, drugs,toxins, oligonucleotides, immunomodulators, hormones, hormoneantagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesisinhibitors, etc. The therapeutic agents recited here are those agentsthat also are useful for administration separately with a targeteddelivery complex as described above. Therapeutic agents include, forexample, chemotherapeutic drugs such as vinca alkaloids, anthracyclines,gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylatingagents, antibiotics, SN-38, COX-2 inhibitors, antimitotics,anti-angiogenic and pro-apoptotic agents, particularly doxorubicin,methotrexate, taxol, CPT-11, camptothecans, proteosome inhibitors, mTORinhibitors, HDAC inhibitors, tyrosine kinase inhibitors, and others.

Other useful cancer chemotherapeutic drugs include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins,hormones, and the like. Suitable chemotherapeutic agents are describedin REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications. Other suitable chemotherapeuticagents, such as experimental drugs, are known to those of skill in theart.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to an anti-cancer antibody,for example as disclosed in U.S. Pat. No. 7,591,994; and U.S. Ser. No.11/388,032, filed Mar. 23, 2006, the Examples section of each of whichis incorporated herein by reference.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate. Other toxins include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,Cell 47:641 (1986), Goldenberg, C A—A Cancer Journal for Clinicians44:43 (1994), Sharkey and Goldenberg, C A—A Cancer Journal forClinicians 56:226 (2006). Additional toxins suitable for use are knownto those of skill in the art and are disclosed in U.S. Pat. No.6,077,499, the Examples section of which is incorporated herein byreference.

As used herein, the term “immunomodulator” includes a cytokine, alymphokine, a monokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, a transforming growth factor (TGF), TGF-α, TGF-β,insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumornecrosis factor (TNF), TNF-α, TNF-β, a mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21 and IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin, endostatin and LT, and the like.

The targeted delivery complex may be administered with animmunoconjugate comprising one or more radioactive isotopes useful fortreating diseased tissue. Particularly useful therapeutic radionuclidesinclude, but are not limited to ¹¹¹In, ¹⁷¹Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe,⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²¹¹Pb. The therapeutic radionuclide preferably has a decay energyin the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keVfor an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000keV for an alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹²⁶I,¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg,^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd,¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se,²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Pharmaceutically Suitable Excipients

The targeted delivery complexes to be delivered to a subject cancomprise one or more pharmaceutically suitable excipients, one or moreadditional ingredients, or some combination of these. The targeteddelivery complex can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the targeted deliverycomplex is combined in a mixture with a pharmaceutically suitableexcipient. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The targeted delivery complex can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one targeted delivery complex as described herein. Ifthe composition containing components for administration is notformulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused. In certain embodiments, a targeted delivery complex may beprovided in the form of a prefilled syringe or autoinjection pencontaining a sterile, liquid formulation or lyophilized preparation ofantibody (e.g., Kivitz et al., Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims.

Example 1 Preparation of Dock-and-Lock (DNL) Constructs

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibody, antibody fragment,siRNA carrier or other effector moieties. For certain preferredembodiments, the antibodies and carrier moieties of the subject targeteddelivery complex may be produced as fusion proteins comprising either adimerization and docking domain (DDD) or anchoring domain (AD) sequence.Although in preferred embodiments the DDD and AD moieties may be joinedto targeting molecules and siRNA carriers as fusion proteins, theskilled artisan will realize that other methods of conjugation exist,particularly for non-protein siRNA carriers, such as chemicalcross-linking, click chemistry reaction, etc.

The technique is not limiting and any protein or peptide of use may beproduced as an AD or DDD fusion protein for incorporation into a DNLconstruct. Where chemical cross-linking is utilized, the AD and DDDconjugates may comprise any molecule that may be cross-linked to an ADor DDD sequence using any cross-linking technique known in the art. Incertain exemplary embodiments, a dendrimer or other polymeric moietysuch as polyethyleneimine or polyethylene glycol (PEG), may beincorporated into a DNL construct, as described in further detail below.

For different types of DNL constructs, different AD or DDD sequences maybe utilized. Exemplary DDD and AD sequences are provided below.

(SEQ ID NO: 33) DDD1: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 34) DDD2: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 35) AD1 QIEYLAKQIVDNAIQQA (SEQ ID NO: 36) AD2:CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD I and DDD2 comprise the DDDsequence of the human RIIα form of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 37) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 38) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 39) CGFEELAWKIAKMIWSDVFQQGC

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD I). To generate Fab-AD expression vectors, the sequences for thehinge, CH2 and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of Ch1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC) followed by four glycinesand a serine, with the final two codons (GS) comprising a Bam HIrestriction site. The 410 bp PCR amplimer was cloned into the PGEMT® PCRcloning vector (PROMEGA®, Inc.) and clones were screened for inserts inthe T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 40) GSGGGGSGGGGSHIQIPPGLTELLOGYTVEVLRQQPPDLVEFAVEYFTRL REARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 41)

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 bp replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 bp digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA 19, hA20 and many others.Generally, the antibody variable region coding sequences were present ina pdHL2 expression vector and the expression vector was converted forproduction of an AD- or DDD-fusion protein as described above. The AD-and DDD-fusion proteins comprising a Fab fragment of any of suchantibodies may be combined, in an approximate ratio of two DDD-fusionproteins per one AD-fusion protein, to generate a trimeric DNL constructcomprising two Fab fragments of a first antibody and one Fab fragment ofa second antibody.

Construction of N-DDD1-Fd-hMN-14-pdHL2

N-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinN-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the aminoterminus of VH via a flexible peptide spacer. The expression vector wasengineered as follows. The DDD1 domain was amplified by PCR.

As a result of the PCR, an NcoI restriction site and the coding sequencefor part of the linker containing a BamHI restriction were appended tothe 5′ and 3′ ends, respectively. The 170 bp PCR amplimer was clonedinto the pGemT vector and clones were screened for inserts in the T7(5′) orientation. The 194 bp insert was excised from the pGemT vectorwith NcoI and SalI restriction enzymes and cloned into the SV3 shuttlevector, which was prepared by digestion with those same enzymes, togenerate the intermediate vector DDD1-SV3.

The hMN-14 Fd sequence was amplified by PCR. As a result of the PCR, aBamHI restriction site and the coding sequence for part of the linkerwere appended to the 5′ end of the amplimer. A stop codon and EagIrestriction site was appended to the 3′ end. The 1043 bp amplimer wascloned into pGemT. The hMN-14-Fd insert was excised from pGemT withBamHI and EagI restriction enzymes and then ligated with DDD1-SV3vector, which was prepared by digestion with those same enzymes, togenerate the construct N-DDD1-hMN-14Fd-SV3.

The N-DDD1-hMN-14 Fd sequence was excised with XhoI and EagI restrictionenzymes and the 1.28 kb insert fragment was ligated with a vectorfragment that was prepared by digestion of C-hMN-14-pdHL2 with thosesame enzymes. The final expression vector was N-DDD1-Fd-hMN-14-pDHL2.The N-linked Fab fragment exhibited similar DNL complex formation andantigen binding characteristics as the C-linked Fab fragment (notshown).

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 2 Generation of TF1 DNL Construct

A large scale preparation of a DNL construct, referred to as TF1, wascarried out as follows. N-DDD2-Fab-hMN-14 (Protein L-purified) andh679-Fab-AD2 (IMP-291-purified) were first mixed in roughlystoichiometric concentrations in 1 mM EDTA, PBS, pH 7.4. Before theaddition of TCEP, SE-HPLC did not show any evidence of a₂b formation(not shown). Instead there were peaks representing a₄ (7.97 min; 200kDa), a₂ (8.91 min; 100 kDa) and B (10.01 min; 50 kDa). Addition of 5 mMTCEP rapidly resulted in the formation of the a₂b complex asdemonstrated by a new peak at 8.43 min, consistent with a 150 kDaprotein (not shown). Apparently there was excess B in this experiment asa peak attributed to h679-Fab-AD2 (9.72 min) was still evident yet noapparent peak corresponding to either a₂ or a₄ was observed. Afterreduction for one hour, the TCEP was removed by overnight dialysisagainst several changes of PBS. The resulting solution was brought to10% DMSO and held overnight at room temperature.

When analyzed by SE-HPLC, the peak representing a₂b appeared to besharper with a slight reduction of the retention time by 0.1 min to 8.31min (not shown), which, based on our previous findings, indicates anincrease in binding affinity. The complex was further purified byIMP-291 affinity chromatography to remove the kappa chain contaminants.As expected, the excess h679-AD2 was co-purified and later removed bypreparative SE-HPLC (not shown).

TF1 is a highly stable complex. When TF1 was tested for binding to anHSG (IMP-239) sensorchip, there was no apparent decrease of the observedresponse at the end of sample injection. In contrast, when a solutioncontaining an equimolar mixture of both C-DDD1-Fab-hMN-14 andh679-Fab-AD1 was tested under similar conditions, the observed increasein response units was accompanied by a detectable drop during andimmediately after sample injection, indicating that the initially formeda₂b structure was unstable. Moreover, whereas subsequent injection ofWI2 gave a substantial increase in response units for TF1, no increasewas evident for the C-DDD1/AD1 mixture.

The additional increase of response units resulting from the binding ofWI2 to TF1 immobilized on the sensorchip corresponds to two fullyfunctional binding sites, each contributed by one subunit ofN-DDD2-Fab-hMN-14. This was confirmed by the ability of TF1 to bind twoFab fragments of WI2 (not shown). When a mixture containing h679-AD2 andN-DDD1-hMN14, which had been reduced and oxidized exactly as TF1, wasanalyzed by BIAcore, there was little additional binding of WI2 (notshown), indicating that a disulfide-stabilized a₁b complex such as TF1could only form through the interaction of DDD2 and AD2.

Two improvements to the process were implemented to reduce the time andefficiency of the process. First, a slight molar excess ofN-DDD2-Fab-hMN-14 present as a mixture of a₄/a₂ structures was used toreact with h679-Fab-AD2 so that no free h679-Fab-AD2 remained and anya₄/a₂ structures not tethered to h679-Fab-AD2, as well as light chains,would be removed by IMP-291 affinity chromatography. Second, hydrophobicinteraction chromatography (HIC) has replaced dialysis or diafiltrationas a means to remove TCEP following reduction, which would not onlyshorten the process time but also add a potential viral removing step.N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced with 5 mM TCEPfor 1 hour at room temperature. The solution was brought to 0.75 Mammonium sulfate and then loaded onto a Butyl FF HIC column. The columnwas washed with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP.The reduced proteins were eluted from the HIC column with PBS andbrought to 10% DMSO. Following incubation at room temperature overnight,highly purified TF1 was isolated by IMP-291 affinity chromatography (notshown). No additional purification steps, such as gel filtration, wererequired.

Example 3 Generation of TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 4 Production of TF10 Bispecific Antibody

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF10 wasderived from hPAM4, a humanized anti-pancreatic cancer mucin MAb thathas been studied in detail as a radiolabeled MAb (e.g., Gold et al.,Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding component wasderived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG)MAb. The TF10 bispecific ([hPAM4]₂×h679) antibody was produced using themethod disclosed for production of the (anti CEA)₂× anti HSG bsAb TF2,as described above. The TF10 construct bears two humanized PAM4 Fabs andone humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

The skilled artisan will realize that the DNL techniques disclosed abovemay be used to produce complexes comprising any combination ofantibodies, immunoconjugates, siRNA carrier moieties or other effectormoieties that may be attached to an AD or DDD moiety.

Example 5 Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Examples, the IgG andFab fusion proteins shown in Table 2 were constructed and incorporatedinto DNL constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 2 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 6 Sequence Variants for DNL

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL construct comprise the amino acid sequences of AD1, AD2,AD3, DDD1, DDD2, DDD3 or DDD3C as discussed above. However, inalternative embodiments sequence variants of AD and/or DDD moieties maybe utilized in construction of the DNL complexes. For example, there areonly four variants of human PKA DDD sequences, corresponding to the DDDmoieties of PKA RIα, RIIα, RIβ and RIIIβ. The RIIα DDD sequence is thebasis of DDD1 and DDD2 disclosed above. The four human PKA DDD sequencesare shown below. The DDD sequence represents residues 1-44 of RIIα, 1-44of RIIβ, 12-61 of RIα and 13-66 of RIβ. (Note that the sequence of DDD1is modified slightly from the human PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 42)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 43) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 44) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 45) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Can et al., 2001, J Biol Chem 276:17332-38; Altoet al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:33below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

(SEQ ID NO: 33) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:35), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:35. The skilledartisan will realize that in designing sequence variants of the ADsequence, one would desirably avoid changing any of the underlinedresidues, while conservative amino acid substitutions might be made forresidues that are less critical for DDD binding.

AKAP-IS sequence QIEYLAKQIVDNAIQQA (SEQ ID NO: 35)

Gold (2006) utilized crystallography and peptide screening to develop aSuperAKAP-IS sequence (SEQ ID NO:46), exhibiting a five order ofmagnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for Ma were residues 8, 11, 15, 16, 18, 19 and 20 (Gold et al.,2006). It is contemplated that in certain alternative embodiments, theSuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:47-49. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:46,the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 46)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 47)QIEYHAKQIVDHAIHQA (SEQ ID NO: 48) QIEYVAKQIVDHAIHQA (SEQ ID NO: 49)

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ ID NO: 50) AKAP79LLIETASSLVKNAIQLSI (SEQ ID NO: 51) AKAP-Lbc LIEEAASRIVDAVIEQVK(SEQ ID NO: 52) RI-Specific AKAPs AKAPce ALYQFADRFSELVISEAL(SEQ ID NO: 53) RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 54) PV38FEELAWKIAKMIWSDVF (SEQ ID NO: 55) Dual-Specificity AKAPs AKAP7ELVRLSKRLVENAVLKAV (SEQ ID NO: 56) MAP2D TAEEVSARIVQVVTAEAV(SEQ ID NO: 57) DAKAP1 QIKQAAFQLISQVILEAT (SEQ ID NO: 58) DAKAP2LAWKIAKMIVSDVMQQ (SEQ ID NO: 59)

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:60-62. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:60), RIAD (SEQ ID NO:61) and PV-38 (SEQ IDNO:62). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 60) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 61) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 62)

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides are provided in Table 1 of Hundsrucker et al., reproduced inTable 3 below. AKAPIS represents a synthetic RII subunit-bindingpeptide. All other peptides are derived from the RII-binding domains ofthe indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 35) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 63) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 64) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 65) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 66) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 67) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 68) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 69) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 70) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 71) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 72) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 73) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 74) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 75) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 76) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 77) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 78) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 79) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 80)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:35). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 35)

Can et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:33. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. The skilled artisan will realize that indesigning sequence variants of DDD, it would be most preferred to avoidchanging the most conserved residues (italicized), and it would bepreferred to also avoid changing the conserved residues (underlined),while conservative amino acid substitutions may be considered forresidues that are neither underlined nor italicized.

(SEQ ID NO: 33) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNLconstructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL constructs.

Example 7 Antibody-Dendrimer DNL Complex for siRNA

Cationic polymers, such as polylysine, polyethylenimine, orpolyamidoamine (PAMAM)-based dendrimers, form complexes with nucleicacids. However, their potential applications as non-viral vectors fordelivering therapeutic genes or siRNAs remain a challenge. One approachto improve selectivity and potency of a dendrimeric nanoparticle may beachieved by conjugation with an antibody that internalizes upon bindingto target cells.

We synthesized and characterized a novel immunoconjugate, designatedE1-G5/2, which was made by the DNL method to comprise half of ageneration 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to astabilized dimer of Fab derived from hRS7, a humanized antibody that israpidly internalized upon binding to the Trop-2 antigen expressed onvarious solid cancers.

Methods

E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2and hRS7-Fab-DDD2, under mild redox conditions, followed by purificationon a Protein L column. To make AD2-G5/2, we derivatized the AD2 peptidewith a maleimide group to react with the single thiol generated fromreducing a G5 PAMAM with a cystamine core and used reversed-phase HPLCto isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a fusion protein inmyeloma cells, as described in the Examples above.

The molecular size, purity and composition of E1-G5/2 were analyzed bysize-exclusion HPLC, SDS-PAGE, and Western blotting. The biologicalfunctions of E1-G5/2 were assessed by binding to an anti-idiotypeantibody against hRS7, a gel retardation assay, and a DNase protectionassay.

Results

E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak(>90%) flanked by several minor peaks. The three constituents of E1-G5/2(Fd-DDD2, the light chain, and AD2-G5/2) were detected by reducingSDS-PAGE and confirmed by Western blotting. Anti-idiotype bindinganalysis revealed E1-G5/2 contained a population of antibody-dendrimerconjugates of different size, all of which were capable of recognizingthe anti-idiotype antibody, thus suggesting structural variability inthe size of the purchased G5 dendrimer. Gel retardation assays showedE1-G5/2 was able to maximally condense plasmid DNA at a charge ratio of6:1 (+/−), with the resulting dendriplexes completely protecting thecomplexed DNA from degradation by DNase I.

Conclusion

The DNL technique can be used to build dendrimer-based nanoparticlesthat are targetable with antibodies. Such agents have improvedproperties as carriers of drugs, plasmids or siRNAs for applications invitro and in vivo.

Example 8 Maleimide AD2 Conjugate for DNL Dendrimers

The peptide IMP 498 up to and including the PEG moiety was synthesizedon a Protein Technologies PS3 peptide synthesizer by the Fmoc method onSieber Amide resin (0.1 mmol scale). The maleimide was added manually bymixing the β-maleimidopropionic acid NHS ester withdiisopropylethylamine and DMF with the resin for 4 hr. The peptide wascleaved from the resin with 15 mL TFA, 0.5 mL H₂O, 0.5 mLtriisopropylsilane, and 0.5 mL thioanisole for 3 hr at room temperature.The peptide was purified by reverse phase HPLC using H₂O/CH₃CN TFAbuffers to obtain about 90 mg of purified product after lyophilization.

Synthesis of Reduced G5 Dendrimer (G5/2)

The G-5 dendrimer (10% in MeOH, Dendritic Nanotechnologies), 2.03 g,7.03×10⁻⁶ mol was reduced with 0.1426 TCEP.HCl 1:1 MeOH/H₂O (˜4 mL) andstirred overnight at room temperature. The reaction mixture was purifiedby reverse phase HPLC on a C-18 column eluted with 0.1% TFA H₂O/CH₃CNbuffers to obtain 0.0633 g of the desired product after lyophilization.

Synthesis of G5/2 Dendrimer-AD2 Conjugate

The G5/2 Dendrimer, 0.0469 g (3.35×10⁻⁶ mol) was mixed with 0.0124 g ofIMP 498 (4.4×10⁻⁶ mol) and dissolved in 1:1 MeOH/1M NaHCO₃ and mixed for19 hr at room temperature followed by treatment with 0.0751 gdithiothreitol and 0.0441 g TCEP HCl. The solution was mixed overnightat room temperature and purified on a C4 reverse phase HPLC column using0.1% TFA H₂O/CH₃CN buffers to obtain 0.0033 g of material containing theconjugated AD2 and dendrimer as judged by gel electrophoresis andWestern blot.

Example 9 Targeted Delivery of siRNA Using Protamine Linked Antibodies

Summary

RNA interference (RNAi) has been shown to down-regulate the expressionof various proteins such as HER2, VEGF, Raf-1, bcl-2, EGFR and numerousothers in preclinical studies. Despite the potential of RNAi to silencespecific genes, the full therapeutic potential of RNAi remains to berealized due to the lack of an effective delivery system to target cellsin vivo.

To address this critical need, we developed novel DNL constructs havingmultiple copies of human protamine tethered to a tumor-targeting,internalizing hRS7 (anti-Trop-2) antibody for targeted delivery ofsiRNAs in vivo. A DDD2-L-thP 1 module comprising truncated humanprotamine (thP1, residues 8 to 29 of human protamine 1) was produced, inwhich the sequences of DDD2 and thP1 were fused respectively to the N-and C-terminal ends of a humanized antibody light chain (FIG. 1). Thesequence of the truncated hP1 (thP1) is shown below. Reaction ofDDD2-L-thP1 with the antibody hRS7-IgG-AD2 under mild redox conditions,as described in the Examples above, resulted in the formation of anE1-L-thP1 complex (FIG. 1), comprising four copies of thP1 attached tothe carboxyl termini of the hRS7 heavy chains.

tHP1 RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 81)

The purity and molecular integrity of E1-L-thP1 following Protein Apurification were determined by size-exclusion HPLC and SDS-PAGE (notshown). In addition, the ability of E1-L-thP1 to bind plasmid DNA orsiRNA was demonstrated by the gel shift assay (FIG. 2). E1-L-thP1 waseffective at binding short double-stranded oligonucleotides (FIG. 2) andin protecting bound DNA from digestion by nucleases added to the sampleor present in serum (not shown).

The ability of the E1-L-thP1 construct to internalize siRNAs intoTrop-2-expressing cancer cells was confirmed by fluorescence microscopyusing FITC-conjugated siRNA and the human Calu-3 lung cancer cell line(FIG. 3).

Methods

The DNL technique was employed to generate E1-L-thP1 (FIG. 1). The hRS7IgG-AD module (FIG. 1A), constructed as described in the Examples above,was expressed in myeloma cells and purified from the culture supernatantusing Protein A affinity chromatography. The DDD2-L-thP1 module (FIG.1B) was expressed as a fusion protein in myeloma cells and was purifiedby Protein L affinity chromatography. Since the CH3-AD2-IgG modulepossesses two AD2 peptides and each can bind to a DDD2 dimer, with eachDDD2 monomer attached to a protamine moiety, the resulting E1-L-thP1conjugate comprises four protamine groups (FIG. 1C). E1-L-thp1 wasformed in nearly quantitative yield from the constituent modules and waspurified to near homogeneity (not shown) with Protein A.

DDD2-L-thP1 was purified using Protein L affinity chromatography andassessed by size exclusion HPLC analysis and SDS-PAGE under reducing andnonreducing conditions (data not shown). A major peak was observed at9.6 min (not shown). SDS-PAGE showed a major band between 30 and 40 kDain reducing gel and a major band about 60 kDa (indicating a dimeric formof DDD2-L-thP1) in nonreducing gel (not shown). The results of Westernblotting confirmed the presence of monomeric DDD2-L-tP1 and dimericDDD2-L-tP1 on probing with anti-DDD antibodies (not shown).

To prepare the E1-L-thP1, hRS7-IgG-AD2 and DDD2-L-thP1 were combined inapproximately equal amounts and reduced glutathione (final concentration1 mM) was added. Following an overnight incubation at room temperature,oxidized glutathione was added (final concentration 2 mM) and theincubation continued for another 24 h. E1-L-thP1 was purified from thereaction mixture by Protein A column chromatography and eluted with 0.1M sodium citrate buffer (pH 3.5). The product peak was neutralized,concentrated, dialyzed with PBS, filtered, and stored in PBS containing5% glycerol at 2 to 8° C. The composition of E1-L-thP1 was confirmed byreducing SDS-PAGE (not shown), which showed the presence of all threeconstituents (AD2-appended heavy chain, DDD2-L-htP1, and light chain).

The ability of DDD2-L-thP1 (not shown) and E1-L-thP1 (FIG. 2) to bindDNA was evaluated by gel shift assay. DDD2-L-thP1 retarded the mobilityof 500 ng of a linear form of 3-kb DNA fragment in 1% agarose at a molarratio of 6 or higher (not shown). FIG. 2 shows that E1-L-thP1 retardedthe mobility of 250 ng of a linear 200-bp DNA duplex in 2% agarose at amolar ratio of 4 or higher (FIG. 2, lanes d-g), whereas no such effectwas observed for hRS7-IgG-AD2 alone (FIG. 2, lane a). The ability ofE1-L-thP1 to protect bound DNA from degradation by exogenous DNase andserum nucleases was also demonstrated (not shown).

The ability of E1-L-thP1 to promote internalization of bound siRNA wasexamined in the Trop-2 expressing ME-180 cervical cell line (FIG. 3).Internalization of the E1-L-thP1 complex was monitored using FITCconjugated goat anti-human antibodies (FIG. 3, lower left). The cellsalone showed no fluorescence (FIG. 3, upper left). Addition ofFITC-labeled siRNA alone resulted in minimal internalization of thesiRNA (FIG. 3, upper right). Internalization of E1-L-thP1 alone wasobserved in 60 minutes at 37° C. (FIG. 3, lower left). E1-L-thP1 wasable to effectively promote internalization of bound FITC-conjugatedsiRNA (FIG. 3, lower right). E1-L-thP1 (10 ng) was mixed with FITC-siRNA(300 nM) and allowed to form E1-L-thP1-siRNA complexes which were thenadded to Trop-2-expressing Calu-3 cells. After incubation for 4 h at 37°C. the cells were checked for internalization of siRNA by fluorescencemicroscopy (FIG. 3, lower right).

The ability of E1-L-thP1 to induce apoptosis by internalization of siRNAwas examined (FIG. 4). E1-L-thP1 (10 μg) was mixed with varying amountsof siRNA (AllStars Cell Death siRNA, Qiagen, Valencia, Calif.). TheE1-L-thP1-siRNA complex was added to ME-180 cells. After 72 h ofincubation, cells were trypsinized and annexin V staining was performedto evaluate apoptosis (FIG. 4). The Cell Death siRNA alone or E1-L-thP1alone had no effect on apoptosis (FIG. 4). Addition of increasingamounts of E1-L-thP1-siRNA produced a dose-dependent increase inapoptosis (FIG. 4). These results show that E1-L-thP1 could effectivelydeliver siRNA molecules into the cells and induce apoptosis of targetcells.

Conclusions

The DNL technology provides a modular approach to efficiently tethermultiple protamine molecules to the anti-Trop-2 hRS7 antibody resultingin the novel molecule E1-L-thP1.

SDS-PAGE demonstrated the homogeneity and purity of E1-L-thP1. DNaseprotection and gel shift assays showed the DNA binding activity ofE1-L-thP1. E1-L-thP1 internalized in the cells like the parental hRS7antibody and was able to effectively internalize siRNA molecules intoTrop-2-expressing cells, such as ME-180 and Calu-3.

The skilled artisan will realize that the DNL technique is not limitedto any specific antibody or siRNA species. Rather, the same methods andcompositions demonstrated herein can be used to make targeted deliverycomplexes comprising any antibody, any siRNA carrier and any siRNAspecies. The use of a bivalent IgG in targeted delivery complexes wouldresult in prolonged circulating half-life and higher binding avidity totarget cells, resulting in increased uptake and improved efficacy. Thisapproach to siRNA mediated cell death is particularly appropriate fordiseases like pancreatic cancer, where present therapies areineffective. Pancreatic cancers are known to express CEACAM6 and CD74,both of which are associated with the invasiveness of pancreatic cancer.Suppression of either CD74 or CEACAM6 expression via RNAi has beenreported to prevent tumor growth in culture and in experimental animals.

Example 10 Apoptosis of Pancreatic Cancer Using siRNAs Against CD74 andCEACAM6

The siRNAs for CD74 (sc-35023, Santa Cruz Biotechnology, Santa Cruz,Calif.) and CEACAM6 [sense strand 5′-CCGGACAGUUCCAUGUAUAdTdT-3′ (SEQ IDNO:82)], are obtained from commercial sources. Sense and antisensesiRNAs are dissolved in 30 mM HEPES buffer to a final concentration of20 μM, heated to 90° C. for 1 min and incubated at 37° C. for 60 min toform duplex siRNA. The duplex siRNA is mixed with E1-L-thP1 andincubated with BxPC-3 (CEACAM6-siRNA) and Capan2 (CD74-siRNA) cells.After 24 h, the changes in the levels of mRNA for the correspondingproteins are determined by real time quantitative PCR analysis. Thelevels CD74 and CEACAM6 proteins are determined by Western blot analysisand immunohistochemistry. Controls include nonspecific siRNA and thenon-targeting DNL complex 20-L-thP1, which contains a humanizedanti-CD20 antibody (hA20).

The effects of reduced expression of CD74 and CEACAM6 on the growth ofpancreatic cancer cells is determined using the clonogenic assay. About1×10³BxPC-3 cells are plated and treated with E1-L-thP1 carryingCEACAM6-siRNA. Media is changed every 3-4 days and after 14 dayscolonies are fixed with 4% para-formaldehyde solution, stained with 0.5%trypan blue and counted. Similar experiments are performed for Capan2cells using E1-L-thP1 carrying CD74-siRNA. The effect of E1-L-thP1carrying both CEACAM6- and CD74-siRNAs on inhibiting the growth ofBxPC-3 and Capan2 cells is determined. Cell proliferation by the MTSassay is performed.

Two xenograft models are established in female athymic nu/nu mice (5weeks of age, weighing 18-20 g). The subcutaneous model has BxPC-3 (ATCCNo. CRL-1687) and Capan2 (ATCC No. HTB-80) implanted in opposite flanksof each animal with treatment initiated once tumors reach 50 mm³. Theorthotopic model bears only BxPC-3 cells and treatment is started 2weeks after implantation.

For the subcutaneous model, the efficacy of E1-L-thP1 to deliver amixture of CEACAM6- and CD74-siRNAs is assessed and compared to that ofE1-L-thP1 to deliver CEACAM6-, CD74-, or control siRNA individually.Additional controls are saline and the use of 20-L-thP1 instead ofE1-L-thP1 to deliver the specific and control siRNAs. The dosage,schedule, and administration are 150 μg/kg based on siRNA, twice weeklyfor 6 weeks, and via tail vein injection (Table 4). Cells are expandedin tissue culture, harvested with Trypsin/EDTA, and re-suspended withmatrigel (1:1) to deliver 5×10⁶ cells in 300 μL.

Animals are monitored daily for signs of toxicity and weighed twiceweekly. Tumor dimensions are measured weekly and tumor volumescalculated.

The orthotopic model is set up as follows. Briefly, nude mice areanesthetized and a left lateral abdominal incision is made. The spleenand attached pancreas are exteriorized with forceps. Then 504 of aBxPC-3 cell suspension (2×10⁶ cells) is injected into the pancreas. Thespleen and pancreas are placed back into the abdominal cavity and theincision closed. Therapy begins two weeks after implantation. Mice aretreated systemically with CEACAM6- or control siRNA bound to E1-L-thP1or 20-L-thP1 with the same dosing schedule and route as the subcutaneousmodel. Animals are monitored daily and weighed weekly.

TABLE 4 Subcutaneous model with dual tumors Group (N) TreatmentDose/Schedule Specific Therapy 1 12 E1-L-thP1-CEACAM6- 150 μg/kg i.v.siRNA (twice weekly × 6) 2 12 E1-L-thP1-CD74-siRNA 150 μg/kg i.v. (twiceweekly × 6) 3 12 E1-L-thP1-CEACAM6- 150 μg/kg each i.v. siRNA + (twiceweekly × 6) E1-L-thP1-CD74-siRNA Controls 4 12 Saline 100 μL i.v. (twiceweekly × 6) 5 12 20-L-thP1-CEACAM6- 150 μg/kg i.v. siRNA (twice weekly ×6) 6 12 20-L-thP1-CD74-siRNA 150 μg/kg i.v. (twice weekly × 6) 7 12E1-L-thP1-control- 150 μg/kg each i.v. siRNA (twice weekly × 6) 8 1220-L-thP1-control- 150 μg/kg each i.v. siRNA (twice weekly × 6)

The results of the study show that both CEACAM6 and CD74 siRNA areinternalized into pancreatic cancer cells by the E1-L-thP1 DNL constructand induce apoptosis of pancreatic cancer, while the control DNLconstruct with non-targeting anti-CD20 antibody is ineffective to inducesiRNA uptake or cancer cell death.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

1. A targeted delivery complex comprising: a) an antibody orantigen-binding fragment thereof attached to one or more anchoringdomain (AD) moieties from an AKAP protein or to one or more dimerizationand docking domain (DDD) moieties from protein kinase A (PKA); and b)one or more siRNA carrier moieties, each siRNA carrier attached to a DDDmoiety from PKA or to an AD moiety from an AKAP protein; wherein twocopies of the DDD moiety form a dimer and bind to the AD moiety to formthe targeted delivery complex and wherein when the antibody or fragmentis attached to one or more AD moieties, the siRNA carrier is attached toa DDD moiety, or when the antibody or fragment is attached to one ormore DDD moieties, the siRNA carrier is attached to an AD moiety.
 2. Thetargeted delivery complex of claim 1, further comprising one or moresiRNA.
 3. The targeted delivery complex of claim 1, further comprisingone or more anti-sense oligonucleotides.
 4. The targeted deliverycomplex of claim 1, further comprising one or more DNA genes.
 5. Thetargeted delivery complex of claim 2, wherein the targeted deliverycomplex comprises at least two different siRNA.
 6. The targeted deliverycomplex of claim 1, wherein the siRNA carrier moiety is selected fromthe group consisting of a dendrimer, a protamine, a histone,histidine-containing reducible polycation, cationic comb-type copolymer,chitosan-thiamine pyrophosphate, polyethyleneimine and polylysine. 7.The targeted delivery complex of claim 6, wherein the dendrimercomprises a polymer selected from the group consisting of PAMAM,polylysine, polypropyleneimine, polyethyleneimine, polyethyleneglycoland carbosilane.
 8. The targeted delivery complex of claim 1, whereinthe siRNA carrier moiety comprises the amino acid sequence of SEQ IDNO:81.
 9. The targeted delivery complex of claim 1, wherein the ADmoiety has the amino acid sequence selected from the group consisting ofSEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.
 10. Thetargeted delivery complex of claim 1, wherein the DDD moiety has anamino acid sequence selected from the group consisting of SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.
 11. The targeted deliverycomplex of claim 2, wherein the siRNA has a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:82.
 12. Amethod of delivering a siRNA, an anti-sense oligonucleotide or a DNAgene comprising: a) obtaining a targeted delivery complex according toclaim 1; b) attaching at least one siRNA, anti-sense oligonucleotide orDNA gene to the complex; and c) administering the targeted deliverycomplex to a subject.
 13. The method of claim 12, wherein the siRNA oranti-sense oligonucleotide inhibits expression of a disease-relatedgene.
 14. The method of claim 12, wherein the siRNA or anti-senseoligonucleotide inhibits expression of a cancer-related gene.
 15. Themethod of claim 12, wherein the targeted delivery complex comprises atleast two different siRNA.
 16. The method of claim 12, wherein the siRNAcarrier moiety is selected from the group consisting of a dendrimer, aprotamine, a histone, histidine-containing reducible polycation,cationic comb-type copolymer, chitosan-thiamine pyrophosphate,polyethyleneimine and polylysine.
 17. The method of claim 16, whereinthe dendrimer comprises a polymer selected from the group consisting ofPAMAM, polylysine, polypropyleneimine, polyethyleneimine,polyethyleneglycol and carbosilane.
 18. The method of claim 12, whereinthe siRNA carrier moiety comprises the amino acid sequence of SEQ IDNO:81.
 19. The method of claim 12, wherein the AD moiety has the aminoacid sequence selected from the group consisting of SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79 and SEQ ID NO:80.
 20. The method of claim 12,wherein the DDD moiety has an amino acid sequence selected from thegroup consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ IDNO:45.
 21. The method of claim 12, wherein the siRNA has a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ IDNO:82.
 22. A method of treating a disease comprising: a) obtaining atargeted delivery complex according to claim 1; b) attaching at leastone siRNA, anti-sense oligonucleotide or DNA gene to the complex; and c)administering the targeted delivery complex to a subject.
 23. The methodof claim 22, wherein the targeted delivery complex comprises at leasttwo different siRNA.
 24. The method of claim 22, wherein the siRNAcarrier moiety is selected from the group consisting of a dendrimer, aprotamine, a histone, histidine-containing reducible polycation,cationic comb-type copolymer, chitosan-thiamine pyrophosphate,polyethyleneimine and polylysine.
 25. The method of claim 24, whereinthe dendrimer comprises a polymer selected from the group consisting ofPAMAM, polylysine, polypropyleneimine, polyethyleneimine,polyethyleneglycol and carbosilane.
 26. The method of claim 22, whereinthe siRNA carrier moiety comprises the amino acid sequence of SEQ IDNO:81.
 27. The method of claim 22, wherein the AD moiety has the aminoacid sequence selected from the group consisting of SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79 and SEQ ID NO:80.
 28. The method of claim 22,wherein the DDD moiety has an amino acid sequence selected from thegroup consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ IDNO:45.
 29. The method of claim 22, wherein the siRNA has a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ IDNO:82.
 30. The method of claim 22, wherein the disease is selected fromthe group consisting of cancer, autoimmune disease, immune dysfunction,graft-versus-host disease, organ transplant rejection, inflammation,infectious disease, cardiac disease and neurologic disease
 31. Themethod of claim 30, wherein the cancer is selected from the groupconsisting of non-Hodgkin's lymphoma, B-cell acute lymphoid leukemia,B-cell chronic lymphoid leukemia, Burkitt lymphoma, Hodgkin's lymphoma,hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia,T-cell lymphoma, T-cell leukemia, multiple myeloma, glioma,Waldenstrom's macroglobulinemia, carcinoma, melanoma, sarcoma, glioma,skin cancer, oral cancer, colon cancer, stomach cancer, colon cancer,lung cancer, breast cancer, ovarian cancer, prostate cancer, uterinecancer, endometrial cancer, cervical cancer, bladder cancer, pancreaticcancer, bone cancer, liver cancer, kidney cancer and testicular cancer.32. The method of claim 30, wherein the autoimmune disease is selectedfrom the group consisting of acute idiopathic thrombocytopenic purpura,chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, Addison's disease,rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis and fibrosingalveolitis.
 33. The method of claim 22, wherein the disease is cancerand the antibody binds to an antigen selected from the group consistingof carbonic anhydrase IX, alpha-fetoprotein, α-actinin-4, A3, antigenspecific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125,CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5,CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25,CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52,CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, DAM, EGFR,EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor,G250 antigen, GAGE, gp 100, GROB, HLA-DR, HM1.24, human chorionicgonadotropin (HCG), HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1),HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R,IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen,KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF),MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A,MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95,NCA90, mucin, placental growth factor, p53, prostatic acid phosphatase,PSA, PRAME, PSMA, P1GF, IGF, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100,survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tnantigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR,ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b,C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET and anoncogene product.
 34. The method of claim 22, wherein the disease iscancer and the antibody is selected from the group consisting of hR1(anti-IGF-1R), hPAM4 (anti-mucin), hA20 (anti-CD20), hA 19 (anti-CD19),hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9(anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15(anti-CEACAM6), hRS7 (anti-Trop-2), hMN-3 (anti-CEACAM6), Ab124(anti-CXCR4) and Ab 125 (anti-CXCR4).
 35. The method of claim 22,further comprising administering at least one therapeutic agent to thesubject.
 36. The method of claim 35, wherein the therapeutic agent isadministered before, simultaneously with or after the targeted deliverycomplex.
 37. The method of claim 35, wherein the therapeutic agent isselected from the group consisting of a radionuclide, a chemotherapeuticdrug, a toxin, an immunomodulator, a hormone, a hormone antagonist, anenzyme, a photoactive therapeutic agent, an anti-angiogenic agent and apro-apoptotic agent.
 38. A pharmaceutical composition comprising atargeted delivery complex according to claim
 1. 39. A kit comprising atargeted delivery complex according to claim 1.